Ouabain Reaction with Microsomal (Sodium‐plus‐Potassium)‐Activated Adenosinetriphosphatase

Abstract

Microsomal (Na+ + K+)-activated adenosinetriphosphatase from eel electroplax binds ouabain in a slow reaction. The rate of binding is modified by enzyme ligands, including nucleotides, orthosphosphate, p-nitrophenylphosphate, carbamylphosphate, Mg2+, Na+, and K+. The actions of these ligands on the enzyme were studied by measuring their effects on the initial rate of ouabain binding.The stimulatory actions ATP, ADP, and Pi depend on the formation of enzyme -Mg -phospholigand complexes. Free nucleotides and Pi are inhibitory. Two alternative pathways for ouabain binding are confirmed : (a) Na+-stimulated, ATP-dependent, and (b) Na+-inhibited, Pi-dependent. I n addition, it is shown that the Na+-stimulated path may depend on any of the organic phosphate ligands, including p-nitrophenylphosphate, carbamylphosphate and ADP. I n contrast, the Na+-inhibited path is supported only by MgHPO, or free Mg2+. There is evidence for non-identical Na+-binding sites : the "a],., value for sttimulation is 20 mM while that for inhibition is 4 mM. I n the presence of both ATP and Pi, the Na+-potentiated-ATP path is favored. K+ inhibits both pathways reversibly and exhibits competition with Na+ and with MgHPO,.ATP decreases the enzyme affinity for K+ while Na+ plus ATP increase the affinity for K+. Since the Pi enzyme complex is observed only in the absence of both Na+ and K+, it is considered a side path not related to the catalytic cycle.The influences of the ligands on ouabain binding may be related to their effects on the enzyme catalytic cycle. There are two general hypotheses for the ligand effects on ouabain reactivity of (Na+ + K+)-ATPase. One, proposed by Sen et al. in 1969, is based on the formation of phosphorylenzyme. The observations are also consistent with an alternative hypothesis which is proposed as follows: (a) the enzyme reactivity with ouabain is increased by the formation of an enzyme -Mg -phospholigand complex regardless of whether the enzyme is also phosphorylated, (b) Na+-activated enzyme has increased affinity for the organic phosphates, which may be considered as substrates, and decreased affinity for Pi and free Mg2+, and (c) K+-activated enzyme has decreased affinity for all the ligands.

Apparent affinity of the Na/K pump for ouabain in cultured chick cardiac myocytes. Effects of Nai and Ko.

“…These results are in contrast to the finding that ouabain binding to partially purified Na/K-ATPase preparations is independent of Na concentration between 1 and 8 mM (Berlin et al, 1986). However, our results do agree with earlier reports in isolated Na/K-ATPase where ouabain binding was found to depend on Na concentration over a wide range (Siegel and Josephson, 1972;Lindenmayer and Schwartz, 1973).…”

Localization of Na pumps in the tracheal epithelium of the dog.

“…In addition to the inhibition of binding by K, we decided to use other tests to see whether the binding of ouabain was to Na pumps. In isolated Na/K ATPases binding is supported by Mg", Na', and ATP (30,42,44,47), and is also temperature sensitive, with the rate of binding less at lower temperatures (17) . In Table II the effects of Na-free (choline) medium, lowering the incubation temperature to 17°C, and hypoxia induced by bubbling with 95% N2, 5% CO 2 are shown.…”

Evidence for Mg2+-Dependent, Na+ + K+-Activated ATPase and Ca2+-ATPase in the Human Term Placenta

“…This is taken to mean that there is a decrease in the available transport ATPase activity. This reduction may occur because of a decrease in free Mg2+ levels due to complexing with ATP, or to competition between ATP and Mg2+-ATP, or both (16).…”